A Non-Hemadsorbing Live-Attenuated Virus Vaccine Candidate Protects Pigs against the Contemporary Pandemic Genotype II African Swine Fever Virus

African swine fever (ASF) is a highly contagious and severe hemorrhagic transboundary swine viral disease with up to a 100% mortality rate, which leads to a tremendous socio-economic loss worldwide. The lack of safe and efficacious ASF vaccines is the greatest challenge in the prevention and control of ASF. In this study, we generated a safe and effective live-attenuated virus (LAV) vaccine candidate VNUA-ASFV-LAVL3 by serially passaging a virulent genotype II strain (VNUA-ASFV-L2) in an immortalized porcine alveolar macrophage cell line (3D4/21, 50 passages). VNUA-ASFV-LAVL3 lost its hemadsorption ability but maintained comparable growth kinetics in 3D4/21 cells to that of the parental strain. Notably, it exhibited significant attenuation of virulence in pigs across different doses (103, 104, and 105 TCID50). All vaccinated pigs remained healthy with no clinical signs of African swine fever virus (ASFV) infection throughout the 28-day observation period of immunization. VNUA-ASFV-LAVL3 was efficiently cleared from the blood at 14–17 days post-infection, even at the highest dose (105 TCID50). Importantly, the attenuation observed in vivo did not compromise the ability of VNUA-ASFV-LAVL3 to induce protective immunity. Vaccination with VNUA-ASFV-LAVL3 elicited robust humoral and cellular immune responses in pigs, achieving 100% protection against a lethal wild-type ASFV (genotype II) challenge at all tested doses (103, 104, and 105 TCID50). Furthermore, a single vaccination (104 TCID50) provided protection for up to 2 months. These findings suggest that VNUA-ASFV-LAVL3 can be utilized as a promising safe and efficacious LAV candidate against the contemporary pandemic genotype II ASFV.


Introduction
African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread [1].The clinical signs and gross pathological lesions of ASF in swine may vary depending on the virus isolate, infection route, dose, and host characteristics.Acute ASF presents with high fever (up to 42 • C), lethargy, anorexia, and inactivity [2].The causative agent, African swine fever virus (ASFV), is a large, enveloped, double-stranded DNA virus belonging to the Asfivirus genus within the Asfarviridae family.Its genome is approximately 170-194 kilobase pairs (kb) and contains over 150 open reading frames (ORFs), depending on the virus strain [3].Based on the p72 major capsid protein gene (B646L), 24 ASFV genotypes (I-XXIV) have been identified [4].
ASF was first reported in Africa in 1921 and emerged for the first time in Europe in I957 [3,4].In 2007, a highly pathogenic strain emerged in the Caucasus region of the Republic of Georgia and swiftly spread to neighboring countries such as the Russian Federation, Armenia, Ukraine, and Azerbaijan [5,6].From 2014 to 2018, ASFV re-emerged in the European Union, originating from eastern nations to Lithuania, Poland, Latvia, and Estonia, subsequently spreading to Hungary, the Czech Republic, and Romania [7,8].The situation escalated globally when ASF reached China in 2018 and spread swiftly across Asia, including Vietnam [9][10][11][12][13].In 2021, it re-emerged in the Western Hemisphere on the Caribbean Island of Hispaniola (Dominican Republic and Haiti) after nearly 40 years [14,15].ASF has also spread westward across Europe in recent years.After a 40-year absence, the disease re-emerged in Italy in January 2022 [16].The outbreak has continued to spread within the country.In 2023, ASF was confirmed for the first time on domestic pig farms in Bosnia and Herzegovina, Greece, and Croatia [17].The disease has now been reported on every continent except Antarctica, despite control efforts in some regions [6,18,19].
Responding to the ASF pandemic, our research group has intensified efforts towards ASF vaccine development since 2019 [18,26].Here, we report the generation of a nonhemadsorbing LAV candidate from a virulent genotype II ASFV using cell passage, and the evaluation of its safety and efficacy in pigs.

Animals
All animal care and protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Vietnam National University of Agriculture (VNUA-2021/01) and at Kansas State University (IACUC#4845).All animal experiments were conducted strictly adhering to the IACUC protocols.Piglets (6-7 weeks old, female) that tested negative for ASFV and ASFV antibodies were obtained from clean pig farms and used for experiments in this study.Pigs were fed a standard commercial diet.In Vietnam, the pigs were housed in the Animal Biosafety Research Facility of the Faculty of Veterinary Medicine, Vietnam National University of Agriculture.In the United States, the pigs were housed under laboratory biosafety level III agriculture (BSL3-Ag) conditions at the Biosecurity Research Institute (BRI), Kansas State University (KSU).

Virus Passage, Titration, and Replication Test In Vitro
Serial passages of the ASFVs were conducted as previously described with slight modifications [39,40].Briefly, monolayers of 3D4/21 cells at 90% confluency were infected with ASFVs at the multiplicity of infection (MOI) of 0.5.After a 2 h adsorption at 37 • C, the inocula were removed.Cells were rinsed twice with phosphate-buffered saline (PBS, pH 7.2, Thermo Scientific, Waltham, MA, USA), replaced with fresh culture media supplemented with 0.5% dimethyl sulfoxide (DMSO, Millipore Sigma, Lenexa, KS, USA), and incubated for 4 days.Culture supernatant was then harvested, titrated, and passaged onto fresh monolayers at an MOI of 0.5.Virus titers in the supernatants at each passage were titrated in PAMs.Briefly, PAMs were pre-seeded (80-100% confluent) and incubated with 10-fold dilutions of the harvested supernatants.For hemadsorption (HAD) testing, 2% porcine red blood cells were added after a 2 h incubation.After four days of culture, the presence of ASFV was assessed by HAD and cytopathic effects (CPEs) in tissue culture under an inverted microscope.HAD 50 and TCID 50 titers were calculated using the method of Reed and Muench [41].
For testing the in vitro replication characteristics of ASFVs, monolayers of PAMs and 3D4/21 cells at 90% confluency in 24-well culture plates were infected with the viruses at an MOI of 0.5.After a 2 h incubation, the inoculums were removed.Cells were washed and replaced with fresh culture media.Cultures (including cells and culture medium) were collected at 0, 24, 36, 48, 60, 72, 84, and 96 h post-infection (HPIs).The collected cultures were subjected to three freeze-thaw cycles.After centrifuging the cell debris, ASFV titers in the supernatant were tested and calculated as described above.All experiments were performed in duplicate.

ASFV Genome Next-Generation Sequencing
Next-generation sequencing of the ASFV genome was performed as described previously [40].Briefly, ASFV DNA was extracted with the QIAamp DNA minikit (Qiagen, Hilden, Germany).The sequencing library was constructed following the manufacturer's instruction of the NEBNext Ultra DNA Library Prep kit (New England Biolabs, Ipswich, MA, USA), and sequencing was performed with the Illumina NovaSeq 150PE sequencing platform (Illumina, San Diego, CA, USA).After sequencing, primer sequences were removed from raw Illumina reads using bbduk of the BBTools Packages (https: //jgi.doe.gov/data-and-tools/bbtools/, last accessed 31 March 2024).QC reads were assembled de novo using SPAdes [42], polished using Pilon version 1.23 [43], and implemented in Unicycler [44].All the contigs were subjected to BLASTN against the NCBI nucleotide database.Open reading frames (ORFs) were predicted using Prodigal [45] and annotated using Prokka 1.14.6 [46].The single contig with BLASTN hit similarity to an ASFV was aligned with a number of reference genomes using MAFFT v7.450 [47].The pairwise comparison of average nucleotide identity (ANI) between ASFV genomes was accomplished by ANI Calculator [48], which is available at https://www.ezbiocloud.net/tools/ani(last accessed 31 March 2024).Other tools for genomic visualization and classifying multigene family (MGF) proteins in ASFV were geneCo [49] and MGFC [50], respectively.All bioinformatic tools were run with default parameter settings.
To assess the duration of protection afforded by VNUA-ASFV-LAVL3, two pig experiments (a one-month vaccination challenge and a two-month vaccination challenge) were conducted.In a one-month vaccination-challenge experiment, five pigs were vaccinated (i.m., once) with 10 4 TCID 50 VNUA-ASFV-LAVL3, three contact pigs (without vaccination) were added to test the shedding of the virus, and three control pigs were kept in a different room.After one month, all pigs were challenged with 10 3 HAD 50 of wild-type VNUA-ASFV-05L1 strain.
Similar to the one-month vaccination-challenge experiment, in the two-month vaccination-challenge experiment, five pigs were vaccinated (i.m., once) with 10 4 TCID 50 VNUA-ASFV-LAVL3, three contact pigs (without vaccination) were added to test the shedding of the virus, and three control pigs were kept in a different room.After two months, all pigs were challenged with 10 3 HAD 50 of the wild-type VNUA-ASFV-05L1 strain.Blood and serum samples were collected at 0, 30, and 60 days post-vaccination (DPVs), and at various DPCs (0, 3, 5, 7, 9, 11, 14, 17, 21, 25, and 28).The presence of clinical signs (anorexia, depression, fever, purple skin discoloration, staggering gait, diarrhea, and cough), body temperature, and survival rate were monitored daily throughout the experiment.
2.6.Quantitative PCR (qPCR) for ASFV qPCR was used to detect ASFV DNA in oral fluid, rectal swabs, blood, and tissue samples collected from the experimental pigs.DNA extraction was performed using an automated King Fisher Duo Prime DNA/RNA extraction system (Thermo-Fisher Scientific, Waltham, MA, USA) with a MagMAX CORE nucleic acid purification kit (Life Sciences, New York, NY, USA) following the manufacturer's instructions.Subsequently, ASFV DNA was quantified using the Platinum SuperMix-UDG kit (Invitrogen, Waltham, MA, USA) on the CFX Optus 96 Realtime PCR system (BioRad, Hercules, CA, USA).Specific primers and a probe targeting the ASFV p72 gene, developed by Haines et al. [51], were employed.Samples with Ct values < 40 were considered positive for ASFV.

Detection of ASFV-Specific Antibody and Cellular Responses in Pigs
A commercial ASF-blocking ELISA kit (INGEZIM PPA COMPAC 11.PPA.k3,Ingenasa, Madrid, Spain) was used to detect anti-ASFV antibodies in serum samples.The assay was performed following the manufacturer's instructions.Each sample's competition percentage (S/N%) was calculated according to the manufacturer's instructions, with interpretations as follows: ≥50% positive, 40-50% doubtful, and ≤40% negative.

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 6.0 (San Diego, CA, USA).The data from assays for virus titration in cell cultures and blood samples in experimental pigs at different time points and efficacy studies were expressed as the mean log titer ± SD (standard deviation) for each group and analyzed with a Student's t-test.The data for antibody and cellular responses were expressed as mean readings ± SD for each group.The significance of differences between the experimental groups was analyzed with an analysis of variance (ANOVA) followed by Turkey's post-test.For all statistical analyses, p values less than 0.05 were considered statistically significant.
p values less than 0.05 were considered statistically significant.

Vaccination with VNUA-ASFV-LAVL3 Confers Full Protection in Pigs
To evaluate the potential of VNUA-ASFV-LAVL3 as a live-attenuated virus vaccine, pigs vaccinated with different doses of VNUA-ASFV-LAVL3 were challenged intramuscularly with 10 3 HAD50 of the highly virulent wild-type VNUA-ASFV-05L1 strain at 28 DPIs.All the vaccinated groups maintained normal rectal temperatures after the challenge, unlike control pigs whose temperatures rapidly increased to 41.8 °C at 5 DPCs (Figure 4A).The challenge virus was almost undetectable in blood samples of all vaccinated groups.Only one pig in the 10 3 TCID50 dose group showed low viral titer (a Ct value of 37.84) at 7 DPCs (Figure 4B).All vaccinated pigs remained healthy and fully protected against the virulent challenge, achieving 100% survival throughout the 28-day DPC observation.In contrast, the challenge virus was detected in control pigs as early as 3-5 DPCs, rapidly increasing by 5-7 DPCs (Figure 4B).They developed typical ASF clinical signs at 5-9 DPCs and succumbed to death by 8-9 DPCs.VNUA-ASFV-LAVL3 vaccinated pigs showed very low viremia, with Ct values ranging from 34.62 to 37.51 (low dose) between 5 and17 DPIs and from 31.52 to 35.17 (high dose) between 3 and 21 DPIs.This viremia rapidly declined, becoming almost undetectable by 17 DPIs (Figure 3B,D,F).On the other hand, control pigs inoculated with the parental VNUA-ASFV-L2 virus reached peak viremia (Ct value~15) at 7 DPIs, remaining high until death by 8-9 DPIs (Figure 3B,D,F).Additionally, ASFV was undetectable in oral fluid or rectal swabs from vaccinated pigs throughout the 28-day observation period (Supplementary Table S1).

Vaccination with VNUA-ASFV-LAVL3 Confers Full Protection in Pigs
To evaluate the potential of VNUA-ASFV-LAVL3 as a live-attenuated virus vaccine, pigs vaccinated with different doses of VNUA-ASFV-LAVL3 were challenged intramuscularly with 10 3 HAD 50 of the highly virulent wild-type VNUA-ASFV-05L1 strain at 28 DPIs.All the vaccinated groups maintained normal rectal temperatures after the challenge, unlike control pigs whose temperatures rapidly increased to 41.8 • C at 5 DPCs (Figure 4A).The challenge virus was almost undetectable in blood samples of all vaccinated

VNUA-ASFV-LAVL3 Vaccination Prevents Replication of Wild-Type ASFV and Protects against Pathological Lesions in Pigs
To further validate the protective efficacy conferred by VNUA-ASFV-LAVL3 in pigs, we investigated the presence of a virulent challenge strain and its associated pathological lesions in various organs of vaccinated pigs at 28 DPCs and control pigs at 8-9 DPCs.At 28 DPCs, organs (brain, heart, lung, liver, stomach, spleen, kidney, bladder, tonsil, lymph node, and bone marrow) collected from control pigs displayed high ASFV titers as determined by qPCR analysis (Table 2).In contrast, pigs vaccinated with VNUA-ASFV-LAVL3 at various doses (10 3 -10 5 TCID 50 ) showed undetectable levels of ASFV in all examined organs at 28 DPCs (Table 2).These data suggest that VNUA-ASFV-LAVL3 vaccination effectively prevents both viral replication and the development of pathological lesions in pigs challenged with the virulent ASFV strain.
Post-mortem examination of the non-immunized control pigs challenged with the wildtype VNUA-ASFV-05L1 revealed severe gross lesions and pathological signs of acute ASF, as previously described [2,53].These pigs displayed extensive macroscopic lesions, including necrosis and hemorrhage in various organs: mandibular lymph nodes, lungs, mesentery, spleen, renal cortex, pericardium, and myocardium.Additionally, the non-immunized pigs exhibited dark and enlarged spleens, swollen livers and gallbladders, and interstitial pulmonary edema (Figure 5A).Conversely, the pigs vaccinated with VNUA-ASFV-LAVL3 showed no clinical signs, pathological lesions, or abnormalities in any examined organs at necropsy (Figure 5B).
Viruses 2024, 16,1326 12 of 18 Post-mortem examination of the non-immunized control pigs challenged with the wild-type VNUA-ASFV-05L1 revealed severe gross lesions and pathological signs of acute ASF, as previously described [2,53].These pigs displayed extensive macroscopic lesions, including necrosis and hemorrhage in various organs: mandibular lymph nodes, lungs, mesentery, spleen, renal cortex, pericardium, and myocardium.Additionally, the non-immunized pigs exhibited dark and enlarged spleens, swollen livers and gallbladders, and interstitial pulmonary edema (Figure 5A).Conversely, the pigs vaccinated with VNUA-ASFV-LAVL3 showed no clinical signs, pathological lesions, or abnormalities in any examined organs at necropsy (Figure 5B).

Discussion
Live-attenuated vaccines, generated by serial passaging of a virus in cultured cells, have proven highly effective in preventing numerous viral diseases, including smallpox, polio, measles, mumps, and yellow fever.These LAVs elicit robust cellular and humoral immune responses, often providing lifelong protection with minimal dosing (one or two doses) [54,55].Carefully controlling passage numbers during cell culture is crucial to balance viral replication and immune induction for developing cell-passaged LAVs.Excessive passaging can weaken the vaccine by eliminating essential protective genes [23].Through careful cell culture and controlled passage times, we have successfully generated an HAD-positive ASF LAV candidate [26].Here, we report a non-HAD ASF LAV candidate, VNUA-ASFV-LAVL3, developed using the same approach.The cell-adapted VNUA-ASFV-LAVL3 can efficiently infect and replicate in 3D4/21 cells with 0.5% DMSO in the culture medium (Figure 1B).Earlier studies highlighted the potential of non-HAD ASFV strains for LAV development.The naturally attenuated non-HAD ASFV/L60 and Lv17/WB/Rie1 strains displayed significantly lower virulence than their parental virulent HAD strains [55,56].Consistent with these findings, the non-HAD VNUA-ASFV-LAVL3 exhibited highly attenuated properties, showing promise for inducing protective immunity against wild-type genotype II ASFV in pigs (Figures 3-5).
A coordinated host response involving both cellular and humoral immunity is essential for combating ASFV infection [57].Cellular immunity, particularly T cell-mediated responses, plays a pivotal role in controlling viral replication by targeting infected cells [58].ELISPOT assays demonstrated that VNUA-ASFV-LAVL3 induced robust cellular immune responses (ASFV-specific IFN-γ) in pigs.While the precise role of antibodies in ASFV protection remains unclear, their presence is considered crucial for defense.Pigs vaccinated with VNUA-ASFV-LAVL3 at doses of 10 3 , 10 4 , and 10 5 TCID50 developed anti-ASFV antibodies as early as 7 DPVs, consistent with previous findings of antibody detection within 7-14 days post-infection in pigs infected with low or moderately virulent ASFV strains [59][60][61].However, the exact mechanisms of how cellular and humoral immune responses work together in combating ASFV require further investigation.
A potential concern with LAVs is the risk of shedding and potential transmission or reversion to virulence [2,21].In our study, no transmission of VNUA-ASFV-LAVL3 from vaccinated pigs to unvaccinated contact pigs was observed, even when challenged 1 or 2 months post-vaccination.All contact pigs remained negative for ASFV-specific antibodies until the challenge.Furthermore, VNUA-ASFV-LAVL3 DNA was undetectable in the organs of vaccinated pigs (Table 2).Notably, a single dose (10 4 TCID 50 ) of VNUA-ASFV-LAVL3 effectively protected pigs against wild-type ASFV challenge for up to 2 months (Table 3).While this study focused on 6-7-week-old piglets, the results strongly support a further investigation of VNUA-ASFV-LAVL3 as a potential broad-spectrum ASFV vaccine.Future studies will assess the vaccine's efficacy in diverse swine populations, including piglets, sows, pregnant sows, and boars.Additionally, we will evaluate its protective capacity against emerging ASFV variants, such as the increasingly prevalent genotype I and II recombinant strains in Asia [62].

Conclusions
This study successfully developed VNUA-ASFV-LAVL3, a highly safe and efficacious vaccine candidate against genotype II ASFV, using cell passage technology.The vaccine candidate efficiently replicates in the commercially available swine macrophage cell line 3D4/21, eliciting robust humoral and cellular immune responses.Importantly, VNUA-ASFV-LAVL3 provided long-term protection against challenges with a highly virulent genotype II ASFV strain.

Viruses 2024, 16 , 1326 7 of 18 Figure 2 .
Figure 2. The graphical panel highlights the deletion of known genes in the MGF regions of the VNUA-ASFV-LAVL3 strain.This panel displays a schematic representation of the ASFV genome organization.Different colors represent major ORF categories.The dashed lines highlight the deleted region in the VNUA-ASFV-LAVL3 strain.

Figure 2 .
Figure 2. The graphical panel highlights the deletion of known genes in the MGF regions of the VNUA-ASFV-LAVL3 strain.This panel displays a schematic representation of the ASFV genome organization.Different colors represent major ORF categories.The dashed lines highlight the deleted region in the VNUA-ASFV-LAVL3 strain.

Figure 5 .
Figure 5. Pathological lesion findings in organs of the vaccinated and non-vaccinated pigs postchallenge at necropsy.(A) Organs from the unvaccinated control pigs at 9 DPCs.(B) Organs from the pigs vaccinated with the VNUA-ASFV-LAVL3 strain at 28 DPCs.

Figure 5 .
Figure 5. Pathological lesion findings in organs of the vaccinated and non-vaccinated pigs postchallenge at necropsy.(A) Organs from the unvaccinated control pigs at 9 DPCs.(B) Organs from the pigs vaccinated with the VNUA-ASFV-LAVL3 strain at 28 DPCs.

Table 3 .
Long-term protection conferred by a single vaccination of VNUA-ASFV-LAVL3 in experimental pigs.